Ab124822

Protein phosphatase inhibitor and protease inhibitor cocktails were purchased from Sigma–Aldrich. Antibodies for ClpX (ab168338, 1:2000), ClpP (ab124822, 1:1000), VDAC (ab34726, 1:1000), α-Synuclein (ab27766, 1:1000), α-Synuclein (ab138501, 1:3000) and α-Synuclein phosphor S129 (ab168381, 1:1000) were from Abcam. GFP (sc-9996, 1:1000), c-Myc (sc-40, 1:1000), α-Synuclein (sc-12767, 1:1000), Enolase (sc-15343, 1:1000) and HSP60 (sc-13115, 1:2000) were from Santa Cruz Biotechnology. β-Actin (A1978, 1:10000) was from Sigma–Aldrich. TH (MAB318, 1:1000) was from Millipore. ClpP (GTX115070, 1:200) was from Genetex. ClpP (NBP1-89557, 1:200) was from Novus. LonP (15440-1-AP, 1:2000), ERAL1 (11478-1-AP, 1:2000), CHCHD3 (25624-1-AP, 1:1000) and WFS1 (11558-1-AP, 1:1000) were from Proteintech. SOD2 (611580, 1:1000) was from BD bioscience. MFN1 (H00055669-M04, 1:1000) was from Abnova. Sirt3 (5490S, 1:1000) was from cell signaling technology.

Total proteins were extracted from treated cells, and the protein concentration in each extract was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, USA). Next, a 40 μg aliquot of protein from each extract was separated by Tris-glycine SDS-PAGE (4% to 20%), and the protein bands were transferred onto polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, St Louis, USA), which were subsequently blocked with nonfat milk. The membranes were then incubated for 8–10 h with the following primary antibodies: anti-CLPP (ab124822; Abcam, Cambridge, UK), anti-PTEN-induced kinase 1 (PINK1) (ab300623; Abcam), anti-parkin (ab77924; Abcam), anti-HSPA8 (ab265664; Abcam) anti-microtubule-associated protein light chain 3 (LC3B) (ab48394; Abcam), and anti-β-actin antibody (ab8227; Abcam). The primary antibody was then detected after incubation with a secondary antibody conjugated with horseradish peroxidase. The protein signals were visualized with an enhanced chemiluminescence (ECL) kit (Pierce).

Equal amounts of total protein extracted from the intestinal crypt cells, were electrophoresed on 12% polyacrylamide/sodium dodecyl sulfate gel and transferred onto polyvinylidene fluoride membranes. After being blocked for 45 min, the membranes were incubated with primary antibodies for 1 h. Subsequently, membranes were incubated with horseradish peroxidase-coupled secondary antibodies for 1 h after 3 times washing with PBS. Finally, the immunoreactivity was detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA, WBKLS0500). The antibodies used were as follows: anti-HSP60 (Abcam, ab46798, 1:1000), anti-LONP1 (Cell Signal Technology, 28020, 1:1000), anti-CDKN1A/p21 (Abclonal, A2691, 1:100), anti-Phospho-eIF2α-S51 (Abclonal, AP0692, 1:1000), anti-ClpP (Abcam, ab124822, 1:1000), anti-ATF4 (Abcam, ab216839, 1:1000), anti-ATF5(Abcam, ab184923, 1:1000), anti-CHOP(Cell Signal Technology, 2895 S, 1:1000), anti-TOM20 (Proteintech, 11802-1-AP, 1:1000), anti-WNT4 (Bio-Techne, MAB4751, 1:1000), anti-SIRT7 (Abclonal, A22735, 1:1000), anti-Actin (HUABIO, ET1702-67, 1:3000), HRP Conjugated Goat anti-Mouse IgG (HUABIO, HA1006, 1:3000) and HRP Conjugated Goat anti-Rabbit IgG (HUABIO, HA1001,1:3000).

Ovarian cancer cells were fixed for 30 min with 4% polyformaldehyde and then permeabilized for 10 min with 0.6% Triton X-100 before being blocked with goat serum. Next, following an overnight incubation at 4°C with anti-CLPP (ab124822; Abcam) or anti-LC3B (ab48394; Abcam) antibody, the cells were incubated for 1 h in the dark with a secondary antibody conjugated to fluorescein isothiocyanate (FITC; Invitrogen). Next, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) staining solution (ab104139; Abcam) at ambient temperature. Finally, the cells were imaged with a fluorescence laser microscope (Olympus, Tokyo, Japan).